Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Assessment of Fungicide Resistance via Molecular Assay in Populations of Podosphaera leucotricha, Causal Agent of Apple Powdery Mildew, in New York.

Podosphaera leucotricha, causal agent of apple powdery mildew, is a pathogen endemic worldwide where apples are produced. In the absence of durable host resistance, the disease is most effectively managed in conventional orchards with single-site fungicides. In New York State, increasingly erratic precipitation patterns and warmer temperatures due to climate change may create a regional environment more conducive to apple powdery mildew development and spread. In this scenario, outbreaks of apple powdery mildew may supplant the apple diseases of current management concern: apple scab and fire blight. Presently, there have been no reports from producers of fungicide control failures for apple powdery mildew, though increased disease incidence has been reported to and observed by the authors. As such, action was needed to assess the fungicide resistance status of populations of P. leucotricha to ensure key classes of single-site fungicides (FRAC 3, demethylation inhibitors, DMI; FRAC 11, quinone outside inhibitors, QoI; and FRAC 7, succinate dehydrogenase inhibitors, SDHI) remain effective. In a 2-year survey (2021 to 2022), we collected 160 samples of P. leucotricha from 43 orchards, representing conventional, organic, low-input, and unmanaged orchards from New York's primary production regions. Samples were screened for mutations in the target genes (CYP51, cytb, and sdhB) historically known to confer fungicide resistance in other fungal pathogens to the DMI, QoI, and SDHI fungicide classes, respectively. Across all samples, no nucleotide sequence mutations that translated into problematic amino acid substitutions were found in the target genes, suggesting that New York populations of P. leucotricha remain sensitive to the DMI, QoI, and SDHI fungicide classes, provided no other fungicide resistance mechanism is at play in the population.

Targeting the vanillic acid decarboxylase gene for Alicyclobacillus acidoterrestris quantification and guaiacol assessment in apple juices using real time PCR.

Alicyclobacillus spp. has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which is characterized by the formation of guaiacol. Previous researches indicate that not all Alicyclobacillus spp. are able to produce guaiacol. The aim of this study was to identify possible differences in the vanillic acid decarboxylase gene involved in guaiacol biosynthesis and then develop specific detection methods for guaiacol producing Alicyclobacillus. Agarose gel electrophoresis results showed that the partial vdcC gene was present in all the guaiacol producing Alicyclobacillus, but absent in non-guaicaol producing strains apart from A. fastidiosus DSM 17978. On the basis of the vdcC gene sequence, a primer pair specific to A. acidoterrestris was designed; then a SYBR Green I real time PCR was established for the direct quantification of A. acidoterrestris in apple juice, and the detection limit was 2.6 × 101 CFU/mL. The developed real time PCR system was used to detect A. acidoterrestris in 36 artificially contaminated apple juice samples and guaiacol production in the sample was also analyzed by GC-MS. The Gompertz model was employed to describe the relationship between A. acidoterrestris cell concentration and guaiacol content, and the value of R2 was 0.854. This work provides an alternative to conventional methods of guaiacol quantification and A. acidoterrestris detection and could be very useful for the early recognition of A. acidoterrestris contamination in fruit juices.

Virulence assessment of Listeria monocytogenes grown in different foods using a Galleria mellonella model.

Various produce including cantaloupe, caramel-coated apples, and packaged salads, have been recognized in recent years as vehicles for listeriosis, a human foodborne disease caused by intracellular pathogen Listeria monocytogenes. Our knowledge regarding the role of these foods in L. monocytogenes virulence, however, is limited. Understanding their role in modulating L. monocytogenes virulence can be useful in risk assessments and for developing control measures. In this study, we employed the Galleria mellonella larvae model to evaluate virulence potential of fifteen clinical, environmental and food isolates of L. monocytogenes, related to three major outbreaks, after growth on different foods. The non-human pathogen Listeria innocua was also included in the panel. Strains were inoculated in parallel in 5ml of brain heart infusion (BHI) broth, and on the surfaces of cantaloupe and apple fragments (5g each) at about 105 colony forming units (CFU)/ml/fragment. One set of inoculated broth and food fragments was incubated at 10°C for 5 days while the second set was kept at 25°C for 3 days. L. monocytogenes cells were recovered from the fruits and BHI, washed twice, re-suspended in saline, and used to inoculate G. mellonella larvae at final concentrations of 106 and 105 CFU/larva. The larvae were incubated at 37°C and monitored for mortality (LT50-time taken to kill 50% of the larvae) and phenotypic changes over seven days. L. monocytogenes grown on cantaloupe and apple flesh surfaces resulted in higher virulence than when grown in BHI. L. monocytogenes infection at 106 CFU/larvae resulted in an average LT50 of ≤ 30, 36 and 47 hours on cantaloupe, apples and BHI, respectively. These results represent a 2.5-4-fold increased mortality compared with an LT50 ≥120 hours in larvae infected with the same doses of L. innocua grown in corresponding matrices. Similar trends were also recorded with doses of about 105 CFU /larvae.

MicroRNA397b negatively regulates resistance of Malus hupehensis to Botryosphaeria dothidea by modulating MhLAC7 involved in lignin biosynthesis.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a vital role in the response of plants to pathogens. Although the microRNA397 family has been implicated in physiological processes as an important regulator, little is known about its function in the resistance of plants to pathogens. Here, Malus hupehensis miR397, which was induced by Botryosphaeria dothidea infection, was identified to directly target M. hupehensis Laccase7 (MhLAC7). The expression analysis of mature Mh-miR397 and MhLAC7 revealed their partly opposite expression patterns. The coexpression of Mh-miR397b in MhLAC7 overexpressing Nicotiana benthamiana suppressed the accumulation of exogenous MhLAC7 and endogenous NbLAC7, which led to decreased lignin content and reduced plant resistance to Botrytis cinerea. As reflected by increasing disease severity and pathogen growth, overexpression of miR397b in both the resistant M. hupehensis and susceptible M. domestica 'Gala' resulted in an increased sensitivity to B. dothidea infection, owing to reduced LAC7 expression and lignin content; however, the inhibition of miR397 had opposite effects. MicroRNA397 functions as a negative regulator in the resistance of Malus to B. dothidea by modulating the LAC7 expression and lignin biosynthesis.

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification.

Most of the molecular diagnostic protocols used for phytoplasmas detection are based on the purification of total nucleic acids and on the use of genomic DNA of the pathogen as the target of amplification. Here we describe a diagnostic approach that, avoiding the purification of nucleic acids and exploiting the amplification of the abundant phytoplasma ribosomal RNA molecules produced during the infectious process, allows reducing the time and the costs necessary for the analysis, without affecting sensitivity and specificity. This is useful in particular when high numbers of analyses are required, as in certification programs, to monitor phytoplasmas classified as quarantine or quality pathogens. The protocol here described can be used for the detection and quantification of Candidatus Phytoplasma mali, Ca. P. pyri, Ca. P. prunorum, Ca. P. vitis, and Ca. P. solani by qPCR, RT-qPCR, ddPCR, and ddRT-PCR techniques based on TaqMan chemistry.

An Electronic Nose for Royal Delicious Apple Quality Assessment - A Tri-layer Approach.

Foodborne pathogens cause serious health issues and have a strong impact on the economy of the country. In this context, quality testing of royal delicious apple by detecting pathogen contamination using an electronic nose, which contains an array of six ready-made sensors, has been proposed. To estimate the types of pathogens, fresh, half and completely contaminated apple samples were considered for bacterial studies. This study revealed the presence of Staphylococcus, Salmonella and Shigella bacteria, which were in the order of zero, 102, 103-104 CFU/mL. Further, the recorded headspace GC-MS spectra of contaminated samples confirmed the presence of bacterial spoilage markers namely acetone, ethyl acetate, ethyl alcohol and acetaldehyde. Voltage swing of 0.2 and 0.5 V was observed for half and completely contaminated apple samples respectively with reference to the fresh sample. Voltage responses of the sensors fed to Principal component analysis and Ward's method of hierarchical cluster algorithms helped to assess the quality of apple samples. By correlating the results of tri-layers namely bacterial count, GCMS data and classification results, reference table was developed and embedded in the ATmega processor of the electronic nose for real-time quality estimation of apple samples.

UV Tolerance of Spoilage Microorganisms and Acid-Shocked and Acid-Adapted Escherichia coli in Apple Juice Treated with a Commercial UV Juice-Processing Unit.

The enhanced thermal tolerance and survival responses of Escherichia coli O157:H7 in acid and acidified foods is a major safety concern for the production of low-pH products, including beverages. Little is known about this phenomenon when using UV light treatments. This study was conducted to evaluate the effects of strain (E. coli O157:H7 strains C7927, ATCC 35150, ATCC 43895, and ATCC 43889 and E. coli ATCC 25922) and physiological state (control-unadapted, acid adapted, and acid shocked) on the UV tolerance of E. coli in apple juice treated under conditions stipulated in current U.S. Food and Drug Administration regulations. A greater than 5-log reduction of E. coli was obtained under all tested conditions. A significant effect of strain (P < 0.05) was observed, but the physiological state did not influence pathogen inactivation (P ≥ 0.05). The UV sensitivity of three spoilage microorganisms (Aspergillus niger, Penicillium commune, and Alicyclobacillus acidoterrestris) was also determined at UV doses of 0 to 98 mJ/cm(2). Alicyclobacillus was the most UV sensitive, followed by Penicillium and Aspergillus. Because of the nonsignificant differences in UV sensitivity of E. coli in different physiological states, the use of an unadapted inoculum would be adequate to conduct challenge studies with the commercial UV unit used in this study at a UV dose of 14 mJ/cm(2). The high UV tolerance of spoilage microorganisms supports the need to use a hurdle approach (e.g., coupling of refrigeration, preservatives, and/or other technologies) to extend the shelf life of UV-treated beverages.

Assessment of diversity and genetic relationships of Neonectria ditissima: the causal agent of fruit tree canker.

BACKGROUND: Neonectria ditissima is one of the most important fungal pathogens of apple trees, where it causes fruit tree canker. Information about the amount and partitioning of genetic variation of this fungus could be helpful for improving orchard management strategies and for breeding apple cultivars with high levels of genetically determined resistance. In this study single-spore Neonectria isolates originating from both the same and from different perithecia, apple cultivars and apple orchards in Sweden and Belgium, were evaluated for AFLP- and SSR-based genetic similarity and for mating system. RESULTS: Seven SSR loci produced a total of 31 alleles with an average of 4 alleles per locus, while 11 AFLP primer combinations produced an average of 35 fragments per primer combination and 71 % polymorphic fragments. An AFLP-based analysis of molecular variance (AMOVA) revealed that 89 % of the variation was found within orchards and 11 % between orchards. Genetic similarity among the studied isolates was illustrated with a principal coordinate analyseis (PCoA) and a dendrogram. AFLP-based Jaccard's similarity coefficients were the highest when single-ascospore isolates obtained from the same perithecium were compared, medium-high for isolates from different perithecia on the same tree, and lowest when isolates from different trees were compared. CONCLUSIONS: Based on the results of PCoA and AMOVA analysis, isolates from the same or geographically close orchards did not group together. Since AFLP profiles differed also when single-ascospore isolates from the same perithecium were compared, the mating system of N. ditissima is most likely heterothallic.

Do some IPM concepts contribute to the development of fungicide resistance? Lessons learned from the apple scab pathosystem in the United States.

One goal of integrated pest management (IPM) as it is currently practiced is an overall reduction in fungicide use in the management of plant disease. Repeated and long-term success of the early broad-spectrum fungicides led to optimism about the capabilities of fungicides, but to an underestimation of the risk of fungicide resistance within agriculture. In 1913, Paul Ehrlich recognized that it was best to 'hit hard and hit early' to prevent microbes from evolving resistance to treatment. This tenet conflicts with the fungicide reduction strategies that have been widely promoted over the past 40 years as integral to IPM. The authors hypothesize that the approaches used to implement IPM have contributed to fungicide resistance problems and may still be driving that process in apple scab management and in IPM requests for proposals. This paper also proposes that IPM as it is currently practiced for plant diseases of perennial systems has been based on the wrong model, and that conceptual shifts in thinking are needed to address the problem of fungicide resistance.

Selection of process conditions by risk assessment for apple juice pasteurization by UV-heat treatments at moderate temperatures.

The effect of bactericidal UV-C treatments (254 nm) on Escherichia coli O157:H7 suspended in apple juice increased synergistically with temperature up to a threshold value. The optimum UV-C treatment temperature was 55 °C, yielding a 58.9% synergistic lethal effect. Under these treatment conditions, the UV-heat (UV-H55 °C) lethal variability achieving 5-log reductions had a logistic distribution (α = 37.92, ß = 1.10). Using this distribution, UV-H55 °C doses to achieve the required juice safety goal with 95, 99, and 99.9% confidence were 41.17, 42.97, and 46.00 J/ml, respectively, i.e., doses higher than the 37.58 J/ml estimated by a deterministic procedure. The public health impact of these results is that the larger UV-H55 °C dose required for achieving 5-log reductions with 95, 99, and 99.9% confidence would reduce the probability of hemolytic uremic syndrome in children by 76.3, 88.6, and 96.9%, respectively. This study illustrates the importance of including the effect of data variability when selecting operational parameters for novel and conventional preservation processes to achieve high food safety standards with the desired confidence level.

Antagonistic effect of Pseudomonas graminis CPA-7 against foodborne pathogens in fresh-cut apples under simulated commercial conditions.

Recently, we reported that the application of the strain CPA-7 of Pseudomonas graminis, previously isolated from apple, could reduce the population of foodborne pathogens on minimally processed (MP) apples and peaches under laboratory conditions. Therefore, the objective of the present work was to find an antioxidant treatment and a packaging atmosphere condition to improve CPA-7 efficacy in reducing a cocktail of four Salmonella and five Listeria monocytogenes strains on MP apples under simulated commercial processing. The effect of CPA-7 application on apple quality and its survival to simulated gastric stress were also evaluated. Ascorbic acid (2%, w/v) and N-acetyl-l-cysteine (1%, w/v) as antioxidant treatments reduced Salmonella, L. monocytogenes and CPA-7 recovery, meanwhile no reduction was observed with NatureSeal(®) AS1 (NS, 6%, w/v). The antagonistic strain was effective on NS-treated apple wedges stored at 10 °C with or without modified atmosphere packaging (MAP). Then, in a semi-commercial assay, efficacy of CPA-7 inoculated at 10(5) and 10(7) cfu mL(-1) against Salmonella and L. monocytogenes strains on MP apples with NS and MAP and stored at 5 and 10 °C was evaluated. Although high CPA-7 concentrations/populations avoided Salmonella growth at 10 °C and lowered L. monocytogenes population increases were observed at both temperatures, the effect was not instantaneous. No effect on apple quality was detected and CPA-7 did not survived to simulated gastric stress throughout storage. Therefore, CPA-7 could avoid pathogens growth on MP apples during storage when use as part of a hurdle technology in combination with disinfection techniques, low storage temperature and MAP.

Application of proanthocyanidins from peanut skins as a natural yeast inhibitory agent.

UNLABELLED: Proanthocyanidins were extracted from peanut skins and investigated for their antimicrobial activity against Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus in traditional growth media (Sabouraud Dextrose and Maltose broth) and a simulated apple juice beverage. Peanut skins extracts (PSE) were prepared through a multisolvent extraction procedure. The PSE extended the lag phase growth of the 3 yeasts studied at a concentration of 1 mg/mL and at 10 mg/mL yeast growth was totally inhibited for 120 h. PSE was fractionated by normal phase high performance liquid chromatography and the active components/fractions were determined. Compounds present in the fractions were identified by liquid chromatography-mass spectrometry to determine the compounds responsible for inhibition. Fractions consisting mostly of A-type proanthocyanidin dimers, trimers, and tetramers showed the highest percent inhibition toward the yeasts tested in this study. Both optical density (OD) and standard enumeration plating methods were performed in this study. The OD method led to an overestimation of the inhibitory effects of PSE, the 2 methods agreed in respect to treatment effects but not the severity of the inhibition. PRACTICAL APPLICATION: There is a growing consumer demand for "fresh like" products containing reduced amounts of chemical preservatives without compromising food safety and quality. Therefore, the goal of this study was to determine if an extract of peanut skins containing flavonoid rich compounds could function as a natural antimicrobial in a model beverage system. Proteins were removed through the process of producing the peanut skin extract, thus it is unlikely to contain peanut allergens. The antimicrobial compounds mentioned in this study were successfully integrated into a model beverage system, and were found to have antimicrobial effect. However, the incorporation of these compounds would likely lead to negative sensory attributes at the concentration needed to achieve an appreciable antimicrobial effect alone.

Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere.

AIMS: Bacterial communities in the apple phyllosphere were examined quantitatively and qualitatively by applying culture-dependent and culture-independent methods. METHODS AND RESULTS: Populations estimated by viewing cells stained with 4',6-diamidino-2-phenylindole generally were at least 100-1000 times greater than populations estimated by culturing on tryptic soy agar (TSA). Of the 44 operational taxonomic units (OTUs; cut-off threshold of 97%) detected in total, five bacterial orders containing 23 OTUs were identified by culturing on TSA, whereas nine orders containing 33 OTUs were identified by 16S rRNA gene cloning of DNA extracted from apple leaf surfaces. Twelve of the 44 OTUs were shared between cultured isolates and 16S rRNA gene clones and included the orders Burkholderiales, Pseudomonadales, Rhizobiales and Sphingomonadales. Three OTUs within the genus Sphingomonas accounted for 40% of isolates and 68% of clones. The Actinomycetales were found only among isolates, whereas the Bacteroidales, Enterobacteriales, Myxococales and Sphingobacteriales were represented in the 16S rRNA gene clone libraries but were absent among isolates. CONCLUSIONS: Culture-independent methods revealed greater numbers and greater richness of bacteria on apple leaves than found by culturing. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to directly compare culture-dependent and independent approaches for assessing bacterial communities in the phyllosphere. The biases introduced by different methods will have a significant impact on studies related to phyllosphere ecology, biological control of plant diseases, reservoirs of antibiotic resistance genes and food safety.

Evaluation of kasugamycin for fire blight management, effect on nontarget bacteria, and assessment of kasugamycin resistance potential in Erwinia amylovora.

The emergence and spread of streptomycin-resistant strains of Erwinia amylovora in Michigan has necessitated the evaluation of new compounds effective for fire blight control. The aminoglycoside antibiotic kasugamycin (Ks) targets the bacterial ribosome and is particularly active against E. amylovora. The efficacy of Ks formulated as Kasumin 2L for control of fire blight was evaluated in six experiments conducted over four field seasons in our experimental orchards in East Lansing, MI. Blossom blight control was statistically equivalent to the industry standard streptomycin in all experiments. E. amylovora populations remained constant on apple flower stigmas pretreated with Kasumin and were ≈100-fold lower than on stigmas treated with water. Kasumin applied to apple trees in the field also resulted in a 100-fold reduced total culturable bacterial population compared with trees treated with water. We performed a prospective analysis of the potential for kasugamycin resistance (Ks(R)) development in E. amylovora which focused on spontaneous resistance development and acquisition of a transferrable Ks(R) gene. In replicated lab experiments, the development of spontaneous resistance in E. amylovora to Ks at 250 or 500 ppm was not observed when cells were directly plated on medium containing high concentrations of the antibiotic. However, exposure to increasing concentrations of Ks in media (initial concentration 25 µg ml(-1)) resulted in the selection of Ks resistance (at 150 µg ml(-1)) in the E. amylovora strains Ea110, Ea273, and Ea1189. Analysis of mutants indicated that they harbored mutations in the kasugamycin target ksgA gene and that all mutants were impacted in relative fitness observable through a reduced growth rate in vitro and decreased virulence in immature pear fruit. The possible occurrence of a reservoir of Ks(R) genes in orchard environments was also examined. Culturable gram-negative bacteria were surveyed from six experimental apple orchards that had received at least one Kasumin application. In total, 401 Ks(R) isolates (42 different species) were recovered from apple flowers and leaves and orchard soil samples. Although we have not established the presence of a transferrable Ks(R) gene in orchard bacteria, the frequency, number of species, and presence of Ks(R) enterobacterial species in orchard samples suggests the possible role of nontarget bacteria in the future transfer of a Ks(R) gene to E. amylovora. Our data confirm the importance of kasugamycin as an alternate antibiotic for fire blight management and lay the groundwork for the development and incorporation of resistance management strategies.

Effect of high pressure CO2 and mild heat processing on natural microorganisms in apple juice.

Apple juice was pasteurized by high pressure carbon dioxide (HPCD) at 20MPa with CO(2) concentration of 4.5-5.3% and mild heat (MH) at atmospheric pressure. Microbial inactivation and stability of natural microorganisms in apple juice were investigated. The temperatures were 37, 42, 47, 52, 57 and 62 degrees C, treatment time was 30min, and storage temperatures of pasteurized apple juice were 2 and 28 degrees C. The aerobic bacteria (AB) treated by MH at 62 degrees C and by HPCD at > or =52 degrees C were almost totally inactivated, the microbial counts were<10CFU/mL. The yeasts and moulds (Y&M) treated by MH at > or =57 degrees C and by HPCD at > or =42 degrees C were totally inactivated. HPCD increased the susceptibility of these natural microorganisms to temperature and enhanced their microbial inactivation. The AB in apple juice treated by HPCD at > or =52 degrees C and the Y&M treated by HPCD at > or =57 degrees C, the AB and the Y&M treated by MH at 62 degrees C showed a better stability during storage at 2 and 28 degrees C, but apple juice treated by HPCD at < or =47 degrees C was characterized with high microbial counts of the AB> or =2.75x10(3)CFU/ml. A viable but non-culturable (VBNC) state of the Y&M treated by MH at 57 degrees C and by HPCD at 42, 47 and 52 degrees C was observed during storage at 28 degrees C. Apparently the proper temperature of HPCD treatment of apple juice at 20MPa and stored at 2 and 28 degrees C was greater than or equal to 52 degrees C, while for MH treatment the proper temperature increased to 62 degrees C in this study.

Safety and quality assessment during the ozonation of cloudy apple juice.

Traditionally, ozone processing within the food industry has focused on solid foods by either gaseous treatment or washing with ozonized water. However, with the FDA's approval of ozone as a direct additive to food, the potential for liquid applications has emerged. This study investigates the effect of ozone processing on microbial inactivation (E. coli ATCC 25922 and NCTC 12900) and quality parameters (color, phenolic content) of cloudy apple juice. Apple juice samples were ozonated at room temperature (20 ± 1.5 °C) with a generated ozone concentration of 0.048 mg O(3) at a constant flow rate of 0.12 L/min and treatment time of 0 to 10 min. E. coli inactivation kinetics in apple juice were described quantitatively by using the Shoulder log-linear and the Weibull model. Ozone treatment of E. coli in apple juice demonstrate that a desired 5 log reduction can be achieved within 5 min. Apple juice color (L*, a*, and b*) and total phenols were significantly affected by ozone concentration and treatment time.

Screen-printed immunosensor modified with carbon nanotubes in a continuous-flow system for the Botrytis cinerea determination in apple tissues.

Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. This paper describes the development of an immunosensor coupled to carbon-based screen-printed electrodes (SPCE) modified with multi-walled carbon nanotubes (CNTs), which show a rapid and sensitive determination of B. cinerea in apple tissues (Red-delicious) using a competitive immunoassay method. Both the infected plant tissue sample and the B. cinerea-specific monoclonal antibody are allowed to react immunologically with the B. cinerea purified antigens immobilized on a rotating disk. Then, the bound antibodies are quantified by a horseradish peroxidise (HRP) enzyme labeled second antibodies specific to mouse IgG, using 4-tertbutylcatechol (4-TBC) as enzymatic mediators. The HRP, in the presence of hydrogen peroxide, catalyses the oxidation of 4-TBC to 4-tertbutyl o-benzoquinone. The electrochemical reduction back to 4-TBC is detected on SPCE-CNT at -0.15 V. The response current is inversely proportional to the amount of the B. cinerea antigens present in the fruit sample. The time consumed per assay was 30 min and the calculated detection limits for electrochemical method and the ELISA procedure are 0.02 and 10 microg mL(-1), respectively. Moreover the intra- and inter-assay coefficients of variation were below 7%. This electrochemical immunosensor promises to be usefully suited to the detection and quantification of B. cinerea in apparently healthy plant prior to the development of the symptoms.

Assessment of the environmental fate of the biological control agent of fire blight, Pseudomonas fluorescens EPS62e, on apple by culture and real-time PCR methods.

The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10(2) cells g (fresh weight)(-1) and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10(7) to 10(8) cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.